The long-term objectives are to understand the path that RNA follows as its synthesis is catalyzed by DNA-dependent RNA polymerase, and to identify the cellular macromolecules that messenger RNA encounters in carrying out its functions. Specific aims are (1) to extend our recently developed photoaffinity labeling techniques to determine all the macromolecules contacted by a growing RNA chain as it passes through a eukaryotic transcription complex (HeLa RNA polymerase II); (2) to study the interactions of prokaryotic (E. coli) transcripts with ribosomes and other components of the translational apparatus; (3) to elucidate the positions on the primary sequence of the E. coli RNA polymerase subunit Sigma that are contacted by the leading end of nascent RNA in transcription complexes; and (4) to design and synthesize new photoaffinity probes as needed for aims 1, 2 and 3. These probes will have different structures, they will differ in chemical reactivity (nitrene, carbene, thiouracil), and they will be incorporated into different nucleotide analogs. Their combined use will provide valuable cross-checks and new information about the environment of RNA during each step of its synthesis. The control of gene expression is important in health and disease, in both human (eukaryotic) and bacterial (prokaryotic) cells. Genes are transcribed by DNA-dependent RNA polymerases. The planned experiments will provide new knowledge of the molecular mechanisms of gene transcription, and of transcription-translation coupling. Better understanding of these basic life processes will ultimately benefit human health.